The invention relates to methods for quenching electrophiles.
The transmission of viral diseases (e.g., hepatitis A, B, and C, acquired immunodeficiency syndrome, and cytomegalovirus infections) by blood or blood products is a significant problem in medicine. Other biological compositions, such as mammalian and hybridoma cell lines, products of cell lines, milk, colostrum, and sperm, can also contain infectious viruses. Screening donor biological compositions for viral markers can help reduce the transmission of viruses to recipients, but many screening methods are directed to only a few discrete viruses, and are therefore incomplete, and may also be less than 100% sensitive. It is therefore important to inactivate viruses contained in donor blood, blood products, or other biological compositions.
A number of agents that are capable of inactivating viruses in blood have been developed. For example, ethyleneimine monomer and ethyleneimine oligomers (including dimers, trimers, and tetramers) are very effective viral inactivating agents. Methods for using ethyleneimine oligomers for inactivating viruses in biological compositions are described in U.S. Ser. No. 09/005,606 (filed Jan. 12, 1998), hereby incorporated by reference. Ethyleneimine oligomers are themselves chemically active, and must therefore be rendered non-reactive before a product, such as blood, is used clinically. Typically, a viral inactivating compound, such as ethyleneimine dimer, is added to a biological composition to inactivate infectious viruses that might be present in the composition. A quenching agent is then added to inactivate the ethyleneimine dimer that remains after viral inactivation has taken place. The end result is a biological composition that is relatively free of infectious viruses, but that is contaminated with quenched inactivating agent and with quenching agent.
In one aspect, the invention features a method of inactivating a contaminant of a biological composition. The method includes the steps of: (a) contacting the biological composition with an inactivating agent including an aziridino moiety or a haloderivative salt thereof, where a portion of the agent reacts with and inactivates the contaminant, and a portion of the agent remains unreacted; (b) contacting the product of step (a) with a quenching agent which includes a quenching moiety under conditions and for a time sufficient to allow the inactivating agent to bond covalently to the quenching moiety; and (c) separating the quenching agent and the quenched inactivating agent from the biological composition.
In a second aspect, the invention features a method of inactivating a contaminant of a biological composition. The method includes the steps of: (a) contacting the biological composition with an inactivating agent including an aziridino moiety or a haloderivative salt thereof, where a portion of the agent reacts with and inactivates the contaminant, and a portion of the agent remains unreacted; (b) contacting the product of step (a) with a quenching moiety, attached to a separation moiety through covalent bonds, under conditions and for a time sufficient to allow the inactivating agent to bond covalently to the quenching moiety; and (c) separating the separation moiety, the quenching moiety and the quenched inactivating agent from the biological composition.
A preferred quenching moiety of the first or second aspect includes a nucleophilic moiety, such as a thiophosphate or thiosulfate moiety; the thiophosphate moiety may be part of an internucleotide linkage of an oligonucleotide sequence.
Inactivating agents of the first or second aspect may be, for example, ethyleneimine, an ethyleneimine oligomer, a haloderivative salt of ethyleneimine, or a haloderivative salt of an ethyleneimine oligomer. A preferred inactivating agent is N-acetylethyleneimine. The biological composition may be selected from the group consisting of whole mammalian blood, purified or partially purified blood proteins, blood cell proteins, milk, saliva, blood plasma, platelet-rich plasma, a plasma concentrate, a precipitate from any fractionation of plasma, a supernatant from any fractionation of plasma, a serum, a cryoprecipitate, a cryosupernatant, a cell lysate, a mammalian cell culture, a mammalian culture supernatant, a placental extract, a product of fermentation, a platelet concentrate, a leukocyte concentrate, semen, red blood cells, and a recombinant protein-containing composition produced in a transgenic mammal. Preferably, the biological composition is whole human blood or human blood plasma. The contaminant may be a virus.
A preferred separation moiety is selected from the group consisting of a bead, a resin, an antibody, and a biotin molecule. The composition of the second aspect preferably also includes a reporter moiety selected from the group consisting of a UV adsorbing moiety and a fluorescent moiety.
In a third aspect, the invention features a method of quenching an electrophile. The method includes contacting the electrophile with a composition including a thiosulfate or thiophosphate moiety attached to a separation moiety, under conditions and for a time sufficient to allow the electrophile to bond covalently to the thiosulfate or thiophosphate moiety.
Preferably, the electrophile includes an aziridino moiety or a haloderivative salt thereof. For example, the electrophile may be ethyleneimine, an ethyleneimine oligomer, a haloderivative salt of ethyleneimine, or a haloderivative salt of an ethyleneimine oligomer. A preferred electrophile is N-acetylethyleneimine. Preferably, the separation moiety is selected from the group consisting of a bead, a resin, an antibody, and a biotin molecule. The composition may further include a reporter moiety, such as a UV adsorbing or fluorescent moiety. The thiophosphate moiety may be part of an internucleotide linkage of an oligonucleotide sequence.
In a fourth aspect, the invention features a method of removing a viral inactivating agent from a biological composition. The method includes the steps of: (a) contacting the inactivating agent with a quenching moiety that is coupled to a separation moiety selected from the group consisting of a bead, a resin, an antibody, and a biotin molecule; and (b) removing the inactivating agent, the quenching moiety, and the separation moiety from the biological composition. Preferably, step (a) includes contacting the inactivating agent with the quenching moiety under conditions and for a time sufficient to allow covalent bonds to form between the inactivating agent and the quenching moiety. A preferred quenching moiety includes a nucleophilic moiety, such as a thiosulfate or thiophosphate moiety.
In a fifth aspect, the invention features a compound which includes (a) a separation moiety; and (b) a thiosulfate or thiophosphate moiety. Preferably, the separation moiety is selected from the group consisting of a bead, a resin, an antibody, and a biotin molecule. The compound may further include a reporter moiety, such as a UV adsorbing or fluorescent group. The thiophosphate moiety may be part of an internucleotide linkage of an oligonucleotide sequence.
By xe2x80x9cbiological compositionxe2x80x9d is meant a composition that contains biological macromolecules, such as proteins, nucleic acids, lipids, and carbohydrates.
By xe2x80x9cquenching moietyxe2x80x9d or xe2x80x9cquenching agentxe2x80x9d is meant a moiety or an agent that is capable of reacting with, and thereby reducing the reactivity of, an electrophilic compound.
By xe2x80x9creporter moietyxe2x80x9d is meant a UV adsorbing or fluorescent group which is added to the quenching agent for the monitoring of removal of the quenching agent and the quenched inactivating agent.
By xe2x80x9cseparation moietyxe2x80x9d is meant a moiety which confers to a compound at least one property which allows for its separation from most other compounds in a biological composition. Preferred properties include selective high-affinity to a compound not normally present in the biological composition, the ability of the moiety to be separated from the biological composition through filtration, centrifugation, or placement in a magnetic field. A bead, a resin, an antibody, and a biotin molecule are each a preferred separation moiety.
The invention provides new methods for the quenching of viral inactivating agents and the subsequent removal of the quenching and inactivating agents from a biological composition. This method results in a biological composition that is relatively free not only of contaminating viruses, but also relatively free of quenched (i.e., non-reactive) inactivating agent and unreacted quenching agent. The invention provides methods which are compatible with methods of removing solvent and detergent from protein-containing preparations which are virally-inactivated by a solvent/detergent method.
Other features and advantages of the invention will be apparent from the following description and from the claims.